Stutz, Cian. The structure of Poz1-Tpz1 reveals a dimerization module in the fission yeast shelterin complex. 2014, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_11106
Here we present the crystal structure of spPoz1_30-249 bound to spTpz1_475-508 at 2.4 Ǻ resolution. Our structure remarkably resembles the structure of the TRF-homology domain (TRFH) of the human shelterin components TRF1 and TRF2. TRFH acts as a dimerization module in human shelterin. We speculated that also Poz1 functions as a dimerization module in fission yeast shelterin, a hypothesis that we subsequently validated by mutational analysis and size-exclusion chromatography. We revealed that Poz1 by itself is monomeric and upon binding of Tpz1 a Poz1-Tpz1 heterotetramer is formed. Furthermore, we showed that also in vivo heterotetramerization is essential for maintaining proper telomere length. Together with the previously reported dimeric existence of Taz1 and Rap1, as well as the recent discovery that Pot1 can dimerize upon telomere binding, these findings suggest an overall dimeric arrangement of the components in the fission yeast shelterin complex.
We replaced Poz1 by the structurally similar human TRF2H-dimerization domain and its binding partner hApollo. While poz1Δ strains show very long telomeres, the TRF2H-Apollo strain showed slow progressive telomere shortening. We suggest that shelterin function was mostly restored because TRF2H-Apollo imitates the primary function of Poz1, which is linking the double-strand and the single-strand binding halves of shelterin complex in a dimeric fashion. Likely, slow telomere shortening is observed because the interactions between Rap1-Poz1-Tpz1 are likely dynamic and regulated, but TRF2H and Apollo were fused to Rap1 and Tpz1 respectively, not allowing association and dissociation, leading to disrupted telomere regulation.
Furthermore, the structure revealed a zinc binding site at the binding interface of Poz1 and Tpz1. Disruption of the zinc site does not affect Poz1-Tpz1 interaction, but leads to decreased solubility of the complex in vitro and long telomeres in vivo. Consequently, binding of zinc is essential for the structural integrity of the complex.
Ultimately, by determining the crystal structure of Poz1_30-249+Tpz1_475-508, we uncovered new details of the architecture of the fission yeast shelterin complex and revealed striking similarities to structural elements found in human shelterin. Furthermore we examined the delicate interplay between the shelterin components and how essential its integrity is for proper telomere length homeostasis.
|Committee Members:||Baumann, Peter|
|Faculties and Departments:||09 Associated Institutions > Friedrich Miescher Institut FMI > Functional organization of the nucleus (Gasser)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||128 p.|
|Last Modified:||30 Jun 2016 10:56|
|Deposited On:||02 Feb 2015 13:20|
Repository Staff Only: item control page